Cloning And Analysis Of The NGX6 Gene Promoter

Research backgroundA novel candidate tumor suppressor gene had been isolated by molecular genetics laboratory of Cancer Research Institute of Central South University with position-candidate cloning strategy and named nasopharyngeal carcinoma associated gene 6(NGX6)(Genbank accession AF188239).Analysis of bioinformation shows that it contains two transmembrane regions.The extracellular region contains one EGF(epidermal growth factor)-like domain and three potential N-glucosylation sites,and the short cytoplasm contains a tyrosine residue that is a potential phosphorylation site by tyrosine kinases.It is obvious down-regulated in nasopharyngeal carcinoma and colorectal carcinoma.Previous study illuminated that NGX6 can not only delay the process of cell cycle,suppress tumor proliferation and implanted neoplastic growth but also suppress the production of tumor vessel; meanwhile it can impair tumor cell migration and invasive ability as well as improve cell adhersion and gap junctional intercellular communication,and can suppress tumor formation in vivo.NGX6 may inhibit Wnt signaling pathway by its negative regulation on beta-catenin and regulate negatively SPAK/JNK signaling pathway by suppressing the nuclear translocation of P-JNK.It down-regulates critical molecule and transcription factor of EGFR-PKC-IKK-NF-κB signaling pathway. NGX6 could also down-regulate the protein level of signal molecules in Ezrin-related invasion pathway.Studies demonstrated that The EGF-like domain and CYTO region were important for NGX6 to regulate cell adhesion.Cytoplasm region was essential for NGX6 to inhibit migration and invasion of tumor cell.The above results suggested that NGX6 play an important role in the growth and development of tumor cell and is associated with migration and invasion of carcinoma. To further reveal the molecular mechanisms of NGX6 gene related with growth,invasion and migration of carcinoma and to illuminate its biological function,in the study,we investigated the transcriptional regulation of NGX6.On the foundation of previous study we expect to identify its specific expressed mechanism.Bioinformatics analysis the transcription regulatory region of NGX6Bioinformatics provide an analysis for 5' flanking region of NGX6 gene.A region spanning from positions -157 to +91 was identified with potential promoter region of NGX6 gene by PromoterInspector program, whereas a region located positions from -257 to +407 was identified with NGX6 promoter by FirstEF program.A CpG island spanning from positions -107 to 299 or -21 to +310 was revealed by CpGplot program and CpGFinder program,respectively.The CpG islands predicted by these two programs overlap with each other,and overlap with NGX6 potential promoter.A region spanning from positions -50 to +50 was identified respectively by TSSW and TRED on-line program.It is common to find several transcription start sites.Searches for binding sites of transcription factors in NGX6 promoter were performed by MatInspector Professional program and TFSEARCH program.No canonical TATA boxes were found,while several CAAT boxes and GC boxes and putative transcription binding sites for Sp1,Egr-1,RREB,P53, MYC-MAX and AP2 were found in NGX6 potential promoter.Cloning and identifying of the promoter of NGX6Using human genomic DNA from human blood cells as templates, a fragment spanning from positions -357 to +769 of NGX6 gene was amplified by PCR.This fragment expresses as strong promoter activity as SV40 promoter.Further analysis of deletion constructs demonstrated that the region spanning from positions -159 to +276 is the proximal promoter of NGX6 gene.The activity of the proximal promoter was identified further by green fluorescent protein bio-assay. The influence of transcriptional factor Sp1 on NGX6The basic mechanism of transcription initiation is the interaction among cis-acting element,trans-acting factor and RNA polymerase.Its essence is the interaction between protein and DNA or protein and protein.It was analyzed that there are several binding sites of transcription factor Sp1,with high Threshold value,in the proximal promoter of NGX6 gene.Mithramycin A,a specific competitor of Sp1, inhibits the promoter activity and endogenous expression of NGX6.To sum up,we have identified NGX6 promoter and ascertained the proximal region.No canonical TATA boxes were found,while several CAAT boxes and GC boxes were discovered in the NGX6 promoter. Inhibition of the Sp1 binding to NGX6 promoter by mithramycin A significantly reduced the promoter activity and the endogenous expression of NGX6 in mRNA level.In order to identify perfectly the function and position of NGX6 during the generation and development of carcinoma,and to provide evidence for the promising clinical of NGX6, we will approach to the effects of DNA methylation on NGX6 promoter and investigate the functions of other cis-acting elements and trans-acting factors of NGX6 promoter.